Biologic drugs, or biologics, such as therapeutic proteins are generally derived from a host cell. For example, a host cell can be genetically engineered to produce a therapeutic recombinant protein. As another example, the biologic can be an endogenous non-recombinant protein. The host cell can be harvested and lysed, and the biologic can be purified from contaminating host cell constituents through a variety of processes. Alternately, the biologic can be secreted by the host cell into conditioned media, the media harvested, and the biologic purified from contaminants in the conditioned media. Host cell contaminants include host cell proteins (HCP).
HCP contaminants can have an undesirable effect even at very low concentration. For example, they can interfere with the function of the biologic. As another example, HCP contaminants can induce an allergic response or rejection of the biologic by the immune system. It is therefore believed that removal of all, substantially all, or most HCP contamination can reduce undesirable effects.
Accordingly, quality control testing of biologics often includes a test to show that HCP contaminants have been sufficiently removed such that HCP contamination is at a low, permissible level. Detection of low level contaminants can be performed using immunochemical assays, which generally have low detection limits. For example, ELISA can be used with a polyclonal antibody mixture that has been developed to have reactivity against a wide range of potential HCPs. Development of the polyclonal antibody mixture requires optimization of the number of potential targets recognized and the sensitivity and specificity of the assay. Polyclonal antibody mixtures thus need to be validated by demonstrating they detect sufficient numbers of HCPs with acceptable sensitivity and specificity prior to their adoption.
HCP assay validation can be performed by two dimensional (2-D) electrophoresis and western blotting. For example, two aliquots of a sample can each be separated by size and isoelectric point on two different 2-D electrophoresis gels. One gel can be stained with a highly sensitive total protein stain such as a silver, zinc, copper, or Coomassie G-250 stain, and the number of stained spots observed to determine an approximate number of total proteins in the sample. The second gel can be transferred to a western blot membrane and probed with a candidate polyclonal antibody mixture. A comparison between the detected proteins via total protein stain and western blot allows the specificity and sensitivity of the antibody mixture to be determined.
Therefore, the technique requires the ability to determine with reasonable confidence whether or not an identified feature/spot between the gel image and the blot image correspond to the same protein. In particular, a match is assumed when a feature is in the same relative position in both the gel and blot images. However, the assignment of a match can be complicated when the images, or individual features within the images, are not in registry with each other. Lack of registry between the gel image and the blot image, or features therein, can occur because of inconsistencies in electrophoresis, transfer, and staining, and the images can be of different size due to shrinkage or swelling of the gel. These sources of inconsistency, and others, can cause incorrect or ambiguous matching.